Immunization with ex vivo-generated dendritic cells (DCs) pulsed with peptide antigen (Ag) or transduced with Ag-expressing recombinant virus represents a promising approach to both the immunotherapy and prevention of HIV/AIDS, other infectious diseases, and cancer. However, DC-based immunization is constrained by some critical limitations, including the limited migration of transferred DCs to secondary lymphoid organs and their rapid elimination in recipients. It is presently unclear whether DC-based vaccines can provide long-lasting protection without the need for frequent re-immunization. In this application, a novel approach to immunotherapy and long-term immunization is proposed based on activation-inducible Ag expression in genetically modified DCs. We hypothesize that: Transplantation with autologous hematopoietic stem cells (HSCs) transduced with lentiviral vector expressing the Ag under the control of a promoter restricting its expression to mature activated DCs results in a state of "controlled by-stander activation" in which the antigen is repeatedly expressed and presented by a limited population of HSC-derived DCs that are currently activated by an ongoing infection or inflammation. This results in a long-term maintenance of high levels of Ag-specific memory T cells. To test this hypothesis, two specific aims are submitted. In Specific Aim 1, the expression pattern of CCL17 promoter will be characterized in mice following the transplantation of transduced HSCs. The tissue distribution of transgene expression, inducibility with different DC stimuli, and the effect of various degrees of myeloablation on the level of chimerism will be studied. In specific Aim 2, the induction and maintenance of Ag-specific T cell responses will be assessed in the recipients of HSCs transduced with lentivirus encoding the OVA Ag under the control of CCL17 promoter. The extent of OVA Ag presentation will be monitored by tetramer staining, intracellular cytokine staining, and by proliferation of adoptively transferred OT-I and OT-II transgenic T cells in vivo. Finally, the presence of protective immune responses will be assessed by a challenge with vaccinia virus expressing OVA Ag. The proposed studies will test a novel approach to the induction of durable immune responses by transplantation of genetically modified HSCs. This strategy offers several critical advantages over the immunization with ex vivo-transduced DCs and, if proven feasible, may be highly instrumental in the immunotherapy of HIV/AIDS, cancer, and other chronic diseases.